Induced pluripotent stem cells (iPSCs) that have been generated from somatic cells have a large variety of current and potential uses in regenerative medicine. Among these uses are generating patient-specific cells, tissues and organs for replacement therapy, and for modeling diseases for research.
Induced pluripotent stem cells have been generated from somatic cells such as fibroblasts derived from a skin biopsy by the overexpression of Yamanaka factors (KLF4, MYC, OCT4 and SOX2) or Thomson/Yu factors (LIN28, NANOG, OCT4 and SOX2). Disadvantageously, however, several weeks are required to prepare cells from a skin biopsy for use in generating induced pluripotent stem cells. Further, induced pluripotent stem cells have also been generated from hematopoietic stem cells (progenitor cells) (HSCs) such as CD34+ cells, CD133+ cells, or from unenriched cells such as mononuclear cells (MNCs) that are harvested from bone marrow, cord blood or peripheral blood, and advantageously do not require substantial time to prepare the cells for use in generating induced pluripotent stem cells. Disadvantageously, however, isolating hematopoietic stem cells or CD34+ cells from mobilized peripheral blood and bone marrow is invasive, time-consuming and has potential risks for the donor. Further, generating induced pluripotent stem cells from cord blood cells has only been accomplished only at an efficiency that is too low for widespread clinical use.
Additionally, in some clinical applications, integration/transgene-free induced pluripotent stem cells are preferably used to ameliorate potential adverse effects due to retroviral or lentiviral integration, or due to the interference by residual expression of reprogramming factors during differentiation of induced pluripotent stem cells into progenies. Several methods have been used to produce integration/transgene-free induced pluripotent stem cells, including the use of adenoviruses, artificial chromosome vectors, the Cre/loxP system or excisable polycistronic lentiviral vectors, minicircle DNA, piggyBac transposon, plasmids, protein transduction, the Sendai virus and synthetic modified mRNA. Disadvantageously, however, these methods are associated with very low efficiency of integration/transgene-free induced pluripotent stem cells generation, require repetitive induction or selection, or require virus production. For example, techniques using excisable polycistronic lentiviral vectors and transposons require a separate step to remove the transgenes once reprogramming has been achieved, while using synthetic modified mRNA to produce integration/transgene-free induced pluripotent stem cells requires the daily addition of mRNA by lipofection, and transfection by lipofection is difficult to achieve with some cell types including blood CD34+ cells.
Further, integration/transgene-free induced pluripotent stem cells have been generated from somatic cells using the Epstein-Barr virus (EBV) latent gene-based episomal vector (EBNA1-based episomal vector) that advantageously requires only one transfection of vector DNA by nucleofection for efficient reprogramming, and that is lost in 5% or more of the cells after each cell division, leading to depletion of the vector from cells after long-term passage. Additionally, integration/transgene-free induced pluripotent stem cells have been generated from somatic cells using the pCEP4 vector (that contains the gene coding for the Epstein Barr nuclear antigen (EBNA1) and OriP sequence). Disadvantageously, however, the use of the Epstein-Barr virus (EBV) latent gene-based episomal vector and pCEP4 vector also requires five to seven additional reprogramming factor genes, including strong oncogenes like Myc (c-Myc) (a regulator gene that codes for a transcription factor) or simian virus 40 large T antigen (SV40LT) that might raise safety concerns for general clinical use of the induced pluripotent stem cells generated by using these factors.
The most cost effective approach for generating integration/transgene-free induced pluripotent stem cells from somatic cells is using EV, a plasmid comprising two elements from Epstein-Bar virus (oriP and EBNA1), because there is no need for packaging of viral vectors and one infection is sufficient for successful reprogramming instead of multiple daily infection or the multiple additions of other factors. Binding of the EBNA1 protein to the virus replicon region oriP maintains a relatively long-term episomal presence of the EV plasmids in mammalian cells. These unique features of EV makes it an ideal vector for generating integration/transgene-free induced pluripotent stem cells. EV yields expression of reprogramming factors at sufficiently high levels for several cell divisions, thus allowing for successful reprogramming after only one infection, while the gradual depletion of plasmids during each cell division leads to the generation of integration/transgene-free induced pluripotent stem cells after approximately 2 months of culture.
Among the various cell types used for reprogramming, fibroblasts from skin biopsy or other sources were initially used in many studies for the generation of iPSCs; however, mononuclear cells (MNCs) from peripheral blood (PB) have been widely accepted as a more convenient and almost unlimited resource for cell reprogramming. Peripheral blood mononuclear cells are a mixed population, containing lymphoid cells, including T cells and B cells, and non-lymphoid cells, including myeloid cells, as well as between 0.01% and 0.1% CD34+ hematopoietic stem/progenitor cells (HSCs). In earlier studies, mature T or B cells were efficiently converted to induced pluripotent stem cells with Sendai virus or EV plasmids. However, induced pluripotent stem cells generated from T cells and B cells contain T cell receptor (TRC) or immunoglobulin (IG) gene rearrangements, restricting their broad applications in regenerative medicine. Therefore, attempts to generate integration/transgene-free induced pluripotent stem cells from non-lymphoid cells have been made, however, these attempts generated only between one and five integration-free induced pluripotent stem cells colonies from 1 ml of peripheral blood which is too low for therapeutic use. More recent approaches using factors including EBNA1 and shRNA against TP53 (also known as p53) generate up to ten induced pluripotent stem cells colonies from 1 ml of peripheral blood in non-T cell culture conditions; however, expression of EBNA1 and TP53 shRNA synergistically inhibits the genome guardian p53, which raises concerns about the genomic integrity of induced pluripotent stem cells generated using this approach.
Therefore, there is a need for a vector and method for generating integration-free induced pluripotent stem cells from somatic cells that are not subject to these disadvantages, where the vector and method generate sufficient numbers of integration/transgene-free induced pluripotent stem cells from somatic cells for therapeutic use in a cost-effective manner that does not require the use of excessive number of factors such as TP53 shRNA.